This invention is in the field of mass spectrometry and instrumentation for the generation of charged droplets, particularly in applications to ion sources for mass spectrometry and related analytical instruments.
Over the last several decades, mass spectrometry has emerged as one of the most broadly applicable analytical tools for detection and characterization of a wide variety of molecules and ions. This is largely due to the extremely sensitive, fast and selective detection provided by mass spectrometric methods. While mass spectrometry provides a highly effective means of identifying a wide class of molecules, its use for analyzing high molecular weight compounds is hindered by problems related to generating, transmitting and detecting gas phase analyte ions of these species.
First, analysis of important biological compounds, such as oligonucleotides and oligopetides, by mass spectrometric methods is severely limited by practical difficulties related to low sample volatility and undesirable fragmentation during vaporization and ionization processes. Importantly, such fragmentation prevents identification of labile, non-covalently bound aggregates of biomolecules, such as proteinxe2x80x94protein complexes and proteinxe2x80x94DNA complexes, that play an important role in many biological systems including signal transduction pathways, gene regulation and transcriptional control. Second, many important biological applications require ultra-high detection sensitivity and resolution that is currently unattainable using conventional mass spectrometric techniques. As a result of these fundamental limitations, the potential for quantitative analysis of samples containing biopolymers remains largely unrealized.
For example, the analysis of complex mixtures of oligonucleotides produced in enzymatic DNA sequencing reactions is currently dominated by time-consuming and labor-intensive electrophoresis techniques that may be complicated by secondary structure. The primary limitation hindering the application mass spectrometry to the field of DNA sequencing is the limited mass range accessible for the analysis of nucleic acids. This limited mass range may be characterized as a decrease in resolution and sensitivity with an increase in ion mass. Specifically, detection sensitivity on the order of 10xe2x88x9215 moles (or 6xc3x97108 molecules) is required in order for mass spectrometric analysis to be competitive with electrophoresis methods and detection sensitivity on the order of 10xe2x88x9218 moles (or 6xc3x97105 molecules) is preferable. Higher resolution is be needed to resolve and correctly identify the DNA fragments in pooled mixtures particularly those resulting from Sanger sequencing reactions.
In addition to DNA sequencing applications, current mass spectrometric techniques lack the ultra high sensitivity required for many other important biomedical applications. For example, the sensitivity needed for single cell analysis of protein expression and post-translational modification patterns via mass spectrometric analysis is simply not currently available. Further, such applications of mass spectrometric analysis necessarily require cumbersome and complex separation procedures prior to mass analysis.
The ability to selectively and sensitively detect components of complex mixtures of biological compounds via mass spectrometry would tremendously aid the advancement of several important fields of scientific research. First, advances in the characterization and detection of samples containing mixtures of oligonucleotides by mass spectrometry would improve the accuracy, speed and reproducibility of DNA sequencing methodologies. In addition, such advances would eliminate problematic interferences arising from secondary structure. Second, enhanced capability for the analysis of complex protein mixtures and multi-subunit protein complexes would revolutionize the use of mass spectrometry in proteomics. Important applications include: protein identification, relative quantification of protein expression levels, identification of protein post-translational modifications, and the analysis of labile protein complexes and aggregates. Finally, advances in mass spectrometric analysis of samples containing complex mixtures of biomolecules would also provide the simultaneous characterization of both high molecular weight and low molecular weight compounds. Detection and characterization of low molecular weight compounds, such as glucose, ATP, NADH, GHT, would aid considerably in elucidating the role of these molecules in regulating a myriad of important cellular processes.
Mass spectrometric analysis involves three fundamental processes: (1) desorption and ionization of a given analyte species to generate a gas phase ion, (2) transmission of the gas phase ion to an analysis region and (3) mass analysis and detection. Although these processes are conceptually distinct, in practice each step is highly interrelated and interdependent. For example, desorption and ionization methods employed to generate gas phase analyte ions significantly influence the transmission and detection efficiencies achievable in mass spectrometry. Accordingly, a great deal of research has been directed toward developing new desorption and ionization methods suitable for the sensitive analysis of high molecular weight compounds.
Conventional ion preparation methods for mass spectrometric analysis have proven unsuitable for high molecular compounds. Vaporization by sublimation or thermal desorption is unfeasible for many high molecular weight species, such as biopolymers, because these compounds tend to have negligibly low vapor pressures. Ionization methods based on the desorption process, however, have proven more effective in generating ions from thermally labile, nonvolatile compounds. Such methods primarily consist of processes that initiate the direct emission of analyte ions from solid or liquid surfaces. Although conventional ion desorption methods, such as plasma desorption, laser desorption, fast particle bombardment and thermospray ionization, are more applicable to nonvolatile compounds, these methods have substantial problems associated with ion fragmentation and low ionization efficiencies for compounds with molecular masses greater than about 2000 Daltons.
To enhance the applicability of mass spectrometry for the analysis of samples containing large molecular weight species, two new ion preparation methods recently emerged: (1) matrix assisted laser desorption and ionization (MALDI) and (2) electrospray ionization (ESI). These methods have profoundly expanded the role of mass spectrometry for the analysis of high molecular weight compounds, such as biomolecules, by providing high ionization efficiency (ionization efficiency=ions formed/molecules consumed in analysis) applicable to a wide range of compounds with molecular weights exceeding 100,000 Daltons. In addition, MALDI and ESI are characterized as xe2x80x9csoftxe2x80x9d desorption and ionization techniques because they are able to both desorb into the gas phase and ionize biomolecules with substantially less fragmentation than conventional ion desorption methods. Karas et. al, Anal. Chem., 60, 2299-2306 (1988) and Karas et. al, Int. J. Mass Spectrom. Ion Proc., 78, 53-68 (1987) describe the application of MALDI as an ion source for mass spectrometry. Fenn, et. al, Science, 246, 64-71 (1989) describes the application of ESI as an ion source for mass spectrometry.
In MALDI mass spectrometry, the analyte of interest is co-crystallized with a small organic compound present in high molar excess relative to the analyte, called the matrix. The MALDI sample, containing analyte incorporated into the organic matrix, is irradiated by a short (xcx9c10 ns) pulse of UV laser radiation at a wavelength resonant with the absorption band of the matrix molecules. The rapid absorption of energy by the matrix causes it to desorb into the gas phase, carrying a portion of the analyte molecules with it. Gas phase proton transfer reactions ionize the analyte molecules within the resultant gas phase plume. Generally, these gas phase proton transfer reactions generate analyte ions in singly and/or doubly charged states. Upon formation, the ions in the source region are accelerated by a high potential electric field, which imparts equal kinetic energy to each ion. Eventually, the ions are conducted through an electric field-free flight tube where they are separated by mass according to their kinetic energies and are detected.
Although MALDI is able to generate gas phase analyte ions from very high molecular weight compounds ( greater than 2000 Daltons), certain aspects of this ion preparation method limit its utility in analyzing complex mixtures of biomolecules. First, fragmentation of analyte molecules during vaporization and ionization gives rise to very complex mass spectra of parent and fragment peaks that are difficult to assign to individual components of a complex mixture. Second, the sensitivity of the technique is dramatically affected by sample preparation methodology and the surface and bulk characteristics of the site irradiated by the laser. As a result, MALDI analysis yields little quantitative information pertaining to the concentrations of the materials analyzed. Finally, the ions generated by MALDI possess a very wide distribution of trajectories due to the laser desorption process, subsequent ionxe2x80x94ion charge repulsion in the plume and collisions with background matrix molecules. This spread in analyte ion trajectories substantially decreases ion transmission efficiencies achievable because only ions translating parallel to the centerline of the mass spectrometer are able to reach the mass analysis region and be detected.
In contrast to MALDI, ESI is a field desorption ionization method that provides a highly reproducible and continuous stream of analyte ions. It is currently believed that the field desorption occurs by a mechanism involving strong electric fields generated at the surface of a charged substrate which extract solute analyte ions from solution into the gas phase. Specifically, in ESI mass spectrometry a solution containing solvent and analyte is passed through a capillary orifice and directed at an opposing plate held near ground. The capillary is maintained at a substantial electric potential (approximately 4 kV) relative to the opposing plate, which serves as the counter electrode. This potential difference generates an intense electric field at the capillary tip, which draws some free ions in the exposed solution to the surface. The electrohydrodynamics of the charged liquid surface causes it to form a cone, referred to as a xe2x80x9cTaylor cone.xe2x80x9d A thin filament of solution extends from this cone until it breaks up into droplets, which carry excess charge on their surface. The result is a stream of small, highly charged droplets that migrate toward the grounded plate. Facilitated by heat and/or the flow of dry bath gases, solvent from the droplets evaporates and the physical size of the droplets decreases to a point where the force due to repulsion of the like charges contained on the surface overcomes the surface tension causing the droplets to fission into xe2x80x9cdaughter droplets.xe2x80x9d This fissioning process may repeat several times depending on the initial size of the parent droplet. Eventually, daughter droplets are formed with a radius of curvature small enough that the electric field at their surface is large enough to desorb analyte species existing as ions in solution. Polar analyte species may also undergo desorption and ionization during electrospray by associating with cations and anions in the liquid sample.
Because ESI generates a highly reproducible stream of gas phase analyte ions directly from a solution containing analyte ions, without the need for complex, off-line sample preparation, it has considerable advantages over analogous MALDI techniques. Certain aspects of ESI, however, currently prevent this ion generating method from achieving its full potential in the analysis complex mixtures of biomolecules. First, as ionization proceeds via the formation of highly charged liquid droplets, ions generated in ESI invariably possess a wide distribution of multiply charged states for each analyte discharged. Accordingly, ESI-MS spectra of mixtures are typically a complex amalgamation of peaks attributable to a large number of populated charged states for every analyte present in the sample. These spectra often possess too many overlapping peaks to permit effective discrimination and identification of the various components of a complex mixture. In addition, highly charged gas phase ions are often unstable and fragment prior to detection, which further increases the complexity of ESI-MS spectra.
Second, a large percentage of ions formed by electrospray ionization are lost during transmission into and through the mass analyzer. Many of these losses can be attributed to divergence in the stream of ions generated. Mutual charge repulsion of ions is a major contributor to beam spreading. In this process, charged droplets and gas phase ions formed by ESI mutually repel each other during transmission from the source to an analysis and detection region. This mutual charge repulsion significantly widens the spatial distribution of the droplet and/or gas phase ion stream and causes significant deviation from the centerline of the mass spectrometer. As the sensitivity of the ESI-MS technique depends strongly on the efficiency with which analyte ions are transported into and through a mass analyzer, the spread in gas phase ion trajectories substantially decreases detection sensitivity attainable in ESI-MS. In addition, spread in ion position is also detrimental to the resolution of the mass determination. For example, in pulsed orthogonal time-of-flight detection, the spread in ion position prior to orthogonal extraction substantially influences the resolution attainable. Divergence of the gas phase ion stream is a major source of deviations in ion start position and, hence, degrades the resolution attainable in the time-of-flight analysis of ions generated by ESI. Typically, small entrances apertures for orthogonal extraction are employed to compensate for these deviations, which ultimately result in a substantial decrease in detection sensitivity.
Finally, ESI, as a continuous ionization source, is not directly compatible with time-of-flight mass analysis. Time-of-flight (TOF) detection is currently the most widely employed detection method for large biomolecules due to its ability to characterize the mass to charge ratio of very high molecular weight compounds. To obtain the benefits from both ESI ion generation and TOF mass analysis, techniques have been developed to segment the continuous ion stream generated in ESI into discrete packets. For example, in conventional TOF analysis electrospray-generated ions are periodically pulsed into an electric field-free-flight tube positioned orthogonal to the axis along which the ions are generated. In the flight tube, the analyte ions are separate by mass according to their kinetic energies and are detected at the end of the flight tube. In this configuration it is essential that the accelerated packets of ions are sufficiently temporally separated with adequate spacing to avoid overlap of consecutive mass spectra. Although ions are generated continuously in ESI-TOF, mass analysis by orthogonal extraction is limited by the duty cycle of the extraction pulse. Most ESI-TOF instruments have a duty cycle between 5% and 50%, depending on the m/z range of the ions being analyzed. Therefore, the majority of ions formed in ESI-TOF are never actually mass analyzed or detected because ion production is not synchronized with detection.
Recently, research efforts have been directed at developing new field desorption ion sources that provide more efficient transmission and detection of the ions generated. One method of improving the transmission and detection efficiencies of ions generated by field desorption involves employing pulsed charged droplet sources that are capable of generating a stream of discrete, single droplets or droplet packets with directed momentum. As the droplets generated by such a droplet source are temporally and spatially separated, mutual charge repulsion between droplets is minimized. Further, ion formation and detection processes may be synchronized by employing a pulsed source, which eliminates the dependence of detection efficiency on the duty cycle of orthogonal extraction in time-of-flight detection.
Although there are a variety of ways that liquid droplets may be generated (e.g. electrical, pneumatic, acoustical or mechanical), a mechanical means of droplet production, piezoelectric droplet generation, has the unique advantage of being able to produce a single droplet event. Piezoelectric droplet generators have been used in many applications including but not limited to ink-jet printing, studies of droplet evaporation and combustion, droplet collision and coalescence, automatic titration, and automated reagent dispensing for molecular biological protocols. Various configurations of piezoelectric droplet sources are described by Zoltan in U.S. Pat. Nos. 3,683,212, 3857,049 and 4,641,155.
There are two piezoeletric methods which produce monodisperse droplets with directed momentum: (1) continuous production by Rayleigh breakup of a liquid jet and (2) droplet-on-demand production by rapid pressure pulsation. In the latter method, a single droplet is released from the end of a capillary as the result of a rapid pressure pulsation generated by a radially contracting piezoelectric element. The size of the droplet produced depends on the solution conditions, orifice diameter, and amplitude and duration of the pressure wave applied. The characteristics of the pressure wave are in turn controlled by the amplitude and duration of the electronic pulse applied to the piezoelectric element.
Hager et al. obtained a mass spectrum of dodecyldiamine (Molecular Mass=201 amu) by incorporating a continuous droplet source with a Sciex TAGA 6000E mass spectrometer (Hager, D.B. et al., Appl. Spectrosc., 46, 1460-1463 (1992)). Using a piezoelectric source, they generated a continuous stream of neutral droplets. After formation, the droplets were charged using an external charging element comprising a corona discharge positioned near the droplet stream. While Hager et al. report successful ion generation via field desorption of droplets generated by a piezoelectric source, electric fields generated by the external corona discharge were observed to significantly perturbed the trajectories of the charged droplets generated. Specifically, FIG. 3 of this reference indicates that the corona discharged caused defection of droplet trajectories up to approximately 450 from the droplets original trajectory. Accordingly, Hager et al. report decreases in ion intensities by a factor of 2-3 relative to conventional electrospray ionization. Further, Hager et al. report no results with higher molecular weight species. Finally, the apparatus described by Hager et al. is not amenable to single droplet production or discretely controlled droplet formation because it employs a continuous droplet source which utilizes Rayleigh breakup of a liquid jet that is not capable of discrete pulsed droplet generation.
Murray and He demonstrated the feasibility of performing mass spectrometry on discretely produced droplets using a MALDI process for generating ions [He, L. And Murray, K., J Mass Spectrom., 34, 909-914 (1999)]. The authors report the use of a piezoelectric droplet source to prepare a sample for MALDI analysis. Specifically, a droplet-on-demand droplet dispenser was used to create dried aerosol particles consisting of matrix and sample. The aerosol particles were ionized by laser irradiation in a MALDI instrument equipped for atmospheric sampling. Murray and He report that 4500 droplets were needed (approximately 50 picomoles of analyte) to obtain a mass spectrum. The authors speculate that the low sensitivity observed was due to poor particle transmission efficiency.
Miliotis et al. report the use of a piezoelectric droplet generator to prepare samples containing an analyte of interest and an organic matrix for MALDI analysis [Miliotis et al., J. Mass Spectrometry, 35, 369-377 (2000)]. Use of the piezoelectric droplet generator in this reference is limited to sample preparation. Miliotis et al. do not report use of a piezoelectric droplet generator as an ion source.
It will be appreciated from the foregoing that a need exists for pulsed field desorption ion sources that are capable of generating a stream of single droplets or discrete, packets of droplets having an electrical charge. The present invention provides a charged droplet source able to provide pulsed production of electrically charged single droplets or discrete packets of electrically charged droplets with directed momentum. Further, this invention describes methods of using this charged droplet source to generate gas phase analyte ions from chemical species, including high molecular weight biopolymers, for detection via conventional mass analysis.
The present invention provides methods and devices for generating charged droplets and/or gas phase ions from liquid samples containing chemical species, including but not limited to chemical species with high molecular mass. The methods and devices of the present invention provide a pulsed stream of electrically charged single droplets or packets of electrically charged droplets of either positive or negative polarity. Further, the methods of the present invention also provide a pulsed stream of single gas phase ions or packets of gas phase analyte ions of either positive or negative polarity. More specifically, the present invention provides charged droplet and/or ion sources with adjustable control of droplet exit time, ion formation time, repetition rate and charge state of the droplets and/or ions formed for use in mass analysis, and particularly in mass spectrometry.
In one embodiment, a charged droplet source of the present invention comprises a piezoelectric droplet generator, which generates discrete and controllable numbers of electrically charged droplets. The droplet source of this embodiment is capable of generating a stream comprising single droplets with momentum substantially directed along a droplet production axis. Alternatively, the droplet source is capable of generating a stream comprising discrete, packets of droplets with momentum substantially directed along a droplet production axis. The droplet generator is capable of providing electrically charged droplets directly and does not require an external charging means. In a preferred embodiment, the charged droplets have a well-characterized spatial distribution along the droplet production axis. The charged droplet source of the present invention is capable of providing a stream of individual droplets and/or packets of droplets that have a substantially uniform and selected spacing along the droplet production axis. Alternatively, the charged droplet source of the present invention is capable of providing a stream of individual droplets and/or packets of droplets in which the spacing between droplets is individually selected and not uniform.
In a specific embodiment, the droplet generator comprises a piezoelectric element with an axial bore having an internal end and an external end. In a preferred embodiment, the piezoelectric element is cylindrical. Within the axial bore is a dispenser element for introducing a liquid sample held at a selected electric potential. The dispenser element has an inlet end that extends a selected distance past the internal end of the axial bore and a dispensing end that extends a select distance past the external end of the axial bore. The external end of the dispensing tube terminates at a small aperture opening, which is positioned directly opposite a grounded element. In a preferred embodiment, the grounded element is metal plate held at a selected electric potential substantially close to ground
The electric potential of the liquid sample is maintained at a selected electric potential by placing the liquid sample in contact with an electrode. The electrode is substantially surrounded by a shield element that substantially prevents the electric field, electromagnetic field or both generated from the electrode from interacting with the piezoelectric element. In a more preferred embodiment, the shield element is the dispenser element itself.
Charged droplets are generated from the liquid sample upon the application of a selected pulsed electric potential to the piezoelectric element, which generates a pulsed pressure wave within the axial bore. In a preferred embodiment, the pulsed pressure wave is a pulsed radially contracting pressure wave. The amplitude and temporal characteristics, including the onset time, frequency, amplitude, rise time and fall time, of the pulsed electric potential is selectively adjustable by a piezoelectric controller operationally connected to the piezoelectric element. In turn, the temporal characteristics and amplitude of the pulsed electric potential control the onset time, frequency, amplitude, rise time fall time and duration of the pressure wave created within the axial bore. The pulsed pressure wave is conveyed through the dispenser element and creates a shock wave in a liquid sample in the dispenser element. This shock wave results in a pressure fluctuation in the liquid sample that generates charged droplets.
The droplet source of the present invention may be operated in two modes with different output: (1) a discrete droplet mode or (2) a pulsed-stream mode. In the discrete droplet mode, each pressure wave results in the formation of an electrically charged single droplet, which exits the dispenser end of the dispenser element. In the pulsed-stream mode, a discrete, elongated stream of electrically charged droplets exits the dispenser end upon application of each pressure wave. In both discrete droplet mode and pulsed-stream mode, the droplet exit time is selectably adjustable by controlling the amplitude and temporal characteristics of the pulsed electric potential applied to the piezoelectric element. Operation of the droplet source of the present invention in the pulsed-stream mode tends to generate smaller charged droplets with a greater ratio of surface area to volume. Droplets with a smaller surface area to volume ratio are especially beneficial when using the charged droplet source of the present invention to generate gas phase ions because these droplets exhibit greater ionization efficiency.
The charged droplet or pulsed stream of droplets exits the dispenser end of the dispenser element at a selected exit time and has a momentum substantially directed along the droplet production axis. Size of the droplets produced from the charged droplet source of the present invention depend on a number of variables including (1) the composition of the liquid sample, (2) the diameter of the small aperture opening, and (3) the amplitude and temporal characteristics of the pulsed electric potential. In another preferred embodiment, the droplet exits the dispensing end into a flow of bath gas that is directed along the droplet production axis. The charged droplets formed may have either positive or negative polarity. Applying a negative electric potential to the electrode in contact with the liquid sample generates negatively charged droplets and applying a positive electric potential to the electrode in contact with the liquid sample generates positively charged droplets.
The piezoelectric element in the present invention may be composed of any material that exhibits piezoelectricity. In an exemplary embodiment, the piezoelectric element is composed of PZT-5A, which is a lead zirconate titanate crystal. In an exemplary embodiment, the piezoelectric element is cylindrical and has a cylindrical axial bore that is oriented along the central axis of the piezoelectric element. Preferably, the piezoelectric cylinder has an outer diameter of about 2.9 millimeters and a length of about 12.7 millimeters. In this preferred embodiment, the cylindrical axial bore has an inner diameter of about 1.7 millimeters. It should be recognized by those skilled in the art, that the piezoelectric element of this invention may have any shape that includes an axial bore and may take on other dimensions than those recited here. Choice of the physical dimensions of the piezoelectric element is important in achieving a pressure wave within the axial bore with the appropriate physical and temporal characteristics.
The dispenser element of the present invention can be made of any material that is capable of transmitting the pressure wave generated by the pulsed pressure wave within the axial bore to the liquid sample. Preferably, the dispensing tube is composed of a chemically inert material that does not substantially conduct electric charge. If an electrically conducting material is chosen, such a stainless steel, an insulator capable of transmitting the pressure wave generated by the pulsed pressure wave is preferably positioned between the dispenser element and the piezoelectric element to substantially prevent electrical conduction from the liquid sample and the piezoelectric element. In preferred embodiments, the dispenser element comprises a glass capillary. In a more preferred embodiment, the dispenser element is a glass capillary with an inner diameter of about 0.8 millimeters and an outer diameter of about 1.5 millimeters. In an exemplary embodiment, the distance the dispensing end of the dispenser element extends from the external end of the axial bore ranges from about 2 millimeters to about 9 millimeters.
It should be understood by persons of ordinary skill in the art that the dispenser element of the present invention may have any shape capable of fitting within the axial bore of the piezoelectric element. In a preferred embodiment, the dispenser element is cylindrical. The dispenser element may also have any volume. A small dispenser element volume may be preferable when analyzing small quantities of liquid sample or low levels of analyte. Alternatively, a large dispenser element volume may be preferable when repeated sampling of a liquid sample in abundance is required.
The dispenser element of the present invention may be bonded into the axial bore of the piezoelectric element or, alternatively, it may be readily removable. If bonded in the axial bore, the adhesive or other bonding material must be capable of transmitting the pulsed pressure wave generated in the axial bore. In a preferred embodiment, the adhesive or other bonding material does not substantially conduct electric charge. In a preferred embodiment, the dispenser element is bonded in the axial bore with epoxy. In another embodiment, the dispenser element is removable to allow external sampling prior to analysis. In this embodiment, the dispenser element may be taken to a sampling site, loaded with sample and returned to the axial bore for droplet formation. In this embodiment, the dispenser element must fit sufficiently tightly within the axial bore to be able to effectively transmit the pressure wave originating from the piezoelectric element.
The small aperture opening of the dispensing end may have any diameter capable of producing charged droplets from the liquid sample upon application of the pulsed electric potential. In a preferred embodiment the small aperture opening has a diameter of about 20 microns or more. A small aperture opening of 20 microns or more is beneficial because it reduces considerably the incidence of tip clogging which is often observed using a small aperture opening below 10 microns in diameter. Further, a 20 micron or greater small aperture opening is desirable because it (1) is easy to clean, (2) is easy to reuse, (3) facilitates sample loading and (4) assists in the initiation of electrospray.
It should be apparent to anyone of skill in the art that any kind of electrode capable of holding the liquid sample at a substantially constant electric potential is useable in the present invention. In preferred embodiments, the electric potential of the liquid sample can be selectively changed. In a preferred embodiment, the electrode is a platinum electrode and the liquid sample is held at a potential ranging from xe2x88x925,000 to 5,000 volts relative to ground and more preferably from xe2x88x923,000 to 3,000 volts relative to ground. Maintaining this lower electric potential generates charged droplets with a lower charge state distribution. A lower charge state distribution may be desirable if the charged droplets are used to generate gas phase ions with minimized fragmentation.
In the charged droplet source of the present invention, the electrode is substantially surrounded by a shield element. The shield element defines a region wherein electric and/or electromagnetic fields generated by the electrode are minimized. In a preferred embodiment the piezoelectric element and/or the piezoelectric controller are within the shielded region. Minimizing the extent of electric fields, electromagnetic fields or both generated from the electrode that interact with the piezoelectric element and/or piezoelectric controller is desirable to allow precise control of the amplitude and temporal characteristics of the pulsed electric potential, the pressure wave and the size and production rate of charged droplets. Accordingly, minimizing the extent electric fields, electromagnetic fields or both generated from the electrode that interact with the piezoelectric element and/or piezoelectric controller is desirable to ensure proper control over the droplet exit time, repetition rate, size and charge state of the droplets. In a preferred embodiment, the dispenser element, itself, is the shield element. In a most preferred embodiment, the dispenser element is a glass capillary that does not substantially conduct electric charge that is cemented into the axial bore using a non-conducting epoxy.
In a preferred embodiment, a plurality of electrically charged droplets is generated sequentially in a flow of bath gas. Each droplet is formed via a separate pressure wave and, therefore, has a unique droplet exit time. The output of this embodiment consists of a stream of individual electrically charged droplets each having a momentum substantially directed along the droplet production axis. This embodiment provides a charged droplet source with controlled timing and spatial location of the droplets along the droplet production axis. In this embodiment, the repetition rate is selectively adjustable. In a more preferred embodiment, a repetition rate is selected that provides a stream of individual drops that are spatially separated such that the individual droplets do not substantially exert forces on each other due to mutual charge repulsion. Minimizing mutual charge repulsion between droplets is desirable because it prevents electrostatic and/or electrodynamic deflection of the droplets from disrupting the well defined droplet trajectories characterized by a momentum substantially directed along the droplet production axis. In another preferred embodiment, the charged droplets have a substantially uniform velocity.
In another embodiment, the electrically charged droplets generated have a substantially uniform diameter. In a preferred embodiment, the electrically charged droplets have a diameter ranging from about 1 micron to about 100 microns. In a more preferred embodiment, the electrically charged droplets have a diameter of about 20 microns. In another embodiment, the composition of the liquid sample, the frequency, amplitude, rise time and fall time of the pressure wave or any combinations thereof are adjusted to select the diameter of the electrically charged droplets formed. In a preferred embodiment, composition of the liquid sample, the frequency, amplitude, rise time and fall time of the pressure wave or any combinations thereof are adjusted to yield droplets having a volume ranging from approximately 1 to about 50 picoliters.
In another embodiment, the charge state of the electrically charged droplets is substantially uniform. In a preferred embodiment, the droplet source of the present invention comprises a source of charged droplets whereby the droplet charging process and the droplet formation process are independently adjustable. This configuration provides independent control of the droplet charge state distribution without substantially influencing the repetition rate, exit time and size of the charged droplets formed. Accordingly, it is possible to limit the degree of droplet charging, independent of droplet size and formation time, as desired by selecting the electric potential applied to the liquid sample. Therefore, the present invention provides a means of producing droplets from liquid samples in which the charge state of individual droplets may be selectively controlled. The ability to select droplet charge state is especially desirable when the droplets generated are used to produce gas phase analyte ions with minimized fragmentation. For this application of the present invention, applying lower electrostatic potentials to the liquid sample is preferred.
In a preferred embodiment, the liquid sample contains chemical species in a solvent, carrier liquid or both. Accordingly, the charged droplets generated also contain chemical species in a solvent, carrier liquid or both. In a preferred embodiment, the chemical species are selected from the group comprising: one or more oligopeptides, one or more oligonucleotides, one or more carbohydrate. In another preferred embodiment, the concentration of the liquid sample is such that each droplet contains a single chemical species in a solvent, carrier liquid or both. In a more preferred embodiment, the concentration of chemical species in the liquid sample ranges from about 1 to 50 picomoles per liter.
Sampling in the present invention may be from a static liquid sample of fixed volume or from a flowing liquid sample. Liquid may be introduced to the dispenser in any manner, including but not limited to (1) filling from the inlet end via application of a positive pressure and (2) aspiration from the dispensing end. In a preferred embodiment, microfluidic sampling methods may be employed by coupling the dispenser element to a microfluidic sampling device. In a preferred embodiment, the dispenser element is operationally coupled to an online purification system to achieve solution phase separation of solutes in a sample containing analytes prior to charged droplet formation. The online purification system may be any instrument or combination of instruments capable of online liquid phase separation. Prior to droplet formation, liquid sample containing solute is separated into fractions, which contain a subset of species (including analytes) of the original solution. For example, separation may be performed so that each analyte is contained in a separate fraction. On line purification methods useful in the present invention include but are not limited to high performance liquid chromatography, capillary electrophoresis, liquid phase chromatography, super critical fluid chromatography, microfiltration methods and flow sorting techniques.
The present invention also comprises an ion source, which generates discrete and controllable numbers of gas phase ions. In a preferred embodiment, the gas phase analyte ions have a momentum substantially directed along a droplet production axis and are spatially distributed along the droplet production axis. In a more preferred embodiment, the gas phase analyte ions generated travel substantially the same well-defined trajectory. An ion source providing gas phase analyte ions that traverse substantially the same trajectory is especially beneficial because it significantly increases the ion collection efficiency attainable.
In this embodiment, the charge droplet source described above is operationally coupled to a field desorption region and the liquid sample contains chemical species in a solvent, carrier liquid or both. In a preferred embodiment, the chemical species are selected from the group comprising: one or more oligopetides, one or more oligonucleotides, one or more and/or one or more carbohydrate. Positively charged droplets or negatively charged droplets of the liquid sample exit the dispenser end of the dispenser element and are conducted by a flow of bath gas through a field desorption region positioned along the droplet production axis. The flow of bath gas can be accomplished by any means capable of providing a flow along the droplet production axis. In the field desorption region, solvent, carrier liquid or both are removed from the droplets by at least partial evaporation or desolvation to produce a flowing stream of smaller charged droplets, gas phase analyte ions or both. In a preferred embodiment, the gas phase analyte ions have a momentum substantially directed along the droplet production axis. Evaporation of positively charged droplets results in formation of gas phase analyte ions that are positively charged and evaporation of negatively charged droplets results in formation of gas phase analyte ions that are negatively charged. The charged droplets, gas phase analyte ions or both remain in the field desorption region for a selected residence time controlled by selectively adjusting the linear flow rate of bath gas and/or the length of the field desorption region. In a preferred embodiment, the charged droplets remain in the field desorption region for a selected residence time sufficient to cause substantially all the chemical species to become gas phase analyte ions. In another preferred embodiment, the gas phase analyte ions have a substantially uniform velocity.
In another embodiment, the rate of evaporation or desolvation in the field desorption region is selectably adjusted. This may be accomplished by methods well known in the art including but not limited to: (1) heating the field desorption region, (2) introducing a flow of dry bath gas to the field desorption region or (3) combinations of these methods with other methods known in the art. Control of the rate of evaporation is beneficial because sufficient evaporation is essential to obtain a high efficiency of ion formation.
In a preferred embodiment of the ion source of the present invention, the field desorption region is substantially free of electric fields generated by sources other than the charged droplets and gas phase analyte ions themselves. In a particular embodiment of the present invention, the electric fields, electromagnetic fields or both generated by the droplet source are substantially minimized in the field desorption region. Maintaining the field desorption region substantially free of electric fields is desirable to prevent disruption of the well-defined trajectories of the gas phase analyte ions generated. In addition minimizing the extent of electric fields, electromagnetic fields or both is beneficial because it prevents unwanted loss of charged droplets and/or ions on the walls of the apparatus and allows for efficient collection of gas phase analyte ions generated by the ion source of the present invention.
Gas phase ions may be prepared from charged droplets generated in either single-droplet or a pulsed-stream mode. Generating gas phase ions from charged droplets generated in the pulsed-stream mode has the advantage that the droplets generated tend to be smaller in diameter and, thus, have large surface area to volume ratios. Higher surface area to volume ratio results in a larger proportion of analyte molecules available for desorption and provides a higher ion production efficiency. Alternatively, generating ions from charged droplets generated in the single-droplet mode has the advantage that mutual charge repulsion of charged droplets is substantially lessened in this mode. Thus, the gas phase ions generated will have a more uniform trajectory.
In a preferred embodiment, individual gas phase analyte ions are generated separately and sequentially in a flow of bath gas. In this embodiment, solution composition is chosen such that each droplet contains only one analyte molecule in a solvent, carrier liquid or both. As each charged droplet is formed via a separate pressure wave, each droplet has a corresponding unique droplet exit time. Upon droplet evaporation in the field desorption region, a single gas phase analyte ion is produced from each charged droplet. In a more preferred embodiment, the repetition rate of the charge droplet source is selected such that it provides a stream of individual gas phase analyte ions that are spatially separated such that the individual analyte ions do not substantially exert forces on each other due to mutual charge repulsion. Minimizing mutual charge repulsion between gas phase analyte ions is beneficial because is preserves the well-defined trajectory of each analyte ion along the droplet production axis.
The present invention also comprises methods of reducing fragmentation of ions generated by field desorption methods. In a preferred embodiment, the ion source of the present invention comprises a source of charged droplets whereby the charging process and the droplet formation process are independently adjustable. This arrangement provides independent control of the droplet charge state attainable without substantially influencing the repetition rate, exit time and size of the charged droplets formed. Selection of the droplet charge state ultimately selects the charge state distribution of gas phase analyte ions formed in the field desorption region. In the present invention it is possible to limit the degree of droplet charging as desired to select a gas phase analyte ion charge state distribution centered around a charge state wherein the gas phase ion is substantially stable and not subject to fragmentation. By employing single droplets produced by a process whereby charging is independent of droplet generation it is possible to limit the degree of droplet charging as desired. Accordingly, the charge state of the droplets generated can be adjusted by selecting the electric potential applied to the liquid sample. This allows for control of the amount of charge on the droplet surface and, hence, the charge state distribution of the gas phase analyte ions generated. Employing lower electric potentials is beneficial because it allows for direct production of gas phase analyte ions in lower charge states, which are less susceptible to fragmentation. Accordingly, the ion source of the present invention is capable of generating gas phase analyte ions with minimized fragmentation. This application of the present invention is especially beneficially for the analysis of labile aggregates and complexes, such as proteinxe2x80x94protein aggregates and protein-DNA aggregates, which fragment easily under high charge state conditions.
Although the ion source of the present invention may be used to generate ions from any chemical species, it is particularly useful for generating ions from high molecular weight compounds, such as peptides, oligonucleotides, carbohydrates, polysaccharides, glycoproteins, lipids and other biopolymers. The methods are generally useful for generating ions from organic polymers. In addition, the ion source of the present invention may be utilized to generate gas phase analyte ions, which possess molecular masses substantially similar to the molecular masses of the parent chemical species from which they are derived while present in the liquid phase. Accordingly, the present invention provides an ion source causing minimal fragmentation to occur during the ionization process. Most preferably for certain applications, the present invention may be utilized to generate gas phase analyte ions with a selectably adjustable charge state distribution.
Alternatively, the ion source of the present invention may be used to induce and control analyte ion fragmentation by selectively varying the extent of multiple charging of the gas phase analyte ions generated. Gas phase ion fragmentation is typically a consequence of the substantially large electric fields generated upon formation of highly multiply charged gas phase analyte ions. The occurrence of controllable fragmentation is useful in determining the identity and structure of chemical species present in liquid samples, the condensed phase and/or the gas phase. The ion source of the present invention may be used to induce fragmentation of gas phase analyte ions by placing the liquid sample in contact with a high electric potential ( greater than 5 kV).
In another embodiment, the ion source of the present invention comprises an ion source without the need for online separation and/or purification of the chemical species prior to gas phase ion formation. In this embodiment, solution conditions are selected such that each charged droplet contains only one chemical species in a solvent, carrier liquid or both. For example, a single analyte ion per charged droplet may be achieved by employing a concentration of less than or equal to about 20 picomoles per liter with a droplet volume of about 10 picoliters. In this embodiment, only one gas phase analyte is released to the gas phase and ionized per charged droplet. As only one ion is formed per droplet, the chemical species in the liquid sample are spatially separated and purified upon ion formation. In another embodiment, a plurality of gas phase analyte ions are generated from each charged droplet. In a preferred embodiment, the output of this embodiment comprises a stream of discrete packets of ions with a momentum substantially directed along the droplet production axis. In this embodiment, solution conditions are selected such that each charged droplet contains a plurality analyte species. Upon at least partial droplet evaporation, a plurality of gas phase analytes is released to the gas phase and ionized.
In a preferred embodiment, the charged droplet source of the present invention is operationally connected to a field desorptionxe2x80x94charge reduction region to provide an ion source with selective control over the charge state distribution of the gas phase ions generated. In this embodiment, the charged droplet source generates a pulsed stream of electrically charged droplets in a flow of bath gas. The stream of charged droplets is conducted through a field desorption charge reduction region where solvent and/or carrier liquid is removed from the droplets by at least partial evaporation to produce a flowing stream of smaller charged droplets and multiply charged gas phase analyte ions. The charged droplets, analyte ions or both remain in the field desorption-charge reduction region for a selected residence time controllable by selectively adjusting the flow rate of bath gas and/or the length of the field desorption region.
Within the field desorptionxe2x80x94charge reduction region, the stream of smaller charged droplets and/or gas phase analyte ions is exposed to electrons and/or gas phase reagent ions of opposite polarity generated from bath gas molecules by a reagent ion source positioned at a selected distance downstream of the electrically charged droplet source. The reagent ion source is surrounded by a shield element for substantially confining the boundaries of electric fields and/or electromagnetic fields generated by the reagent ion source. Electrons, reagent ions or both, generated by the reagent ion source, react with charged droplets, analyte ions or both within at least a portion of the field desorption-charge reduction region and reduce the charge-state distribution of the analyte ions in the flow of bath gas. Accordingly, ionxe2x80x94ion, ionxe2x80x94droplet, electronxe2x80x94ion and/or electronxe2x80x94droplet reactions result in the formation of gas phase analyte ions having a selected charge-state distribution. In a preferred embodiment, the charge state distribution of gas phase analyte ions is selectively adjustable by varying the interaction time between gas phase analyte ions and/or charged droplets and the gas phase reagent ions and/or electrons. In addition, the charge-state of gas phase analyte ions may be controlled by adjusting the rate of production of electrons, reagent ions or both from the reagent ion source. In addition, an ion source of the present invention is capable of generating an output consisting of analyte ions with a charge-state distribution that may be selected or may be varied as a function of time.
In another embodiment, the ion source of the present invention is operationally coupled to a charged particle analyzer capable of identifying, classifying and detecting charged particles. This embodiment provides a method of determining the composition and identity of substances, which may be present in a mixture. In an exemplary embodiment, the ion source of the present invention is operationally coupled to a mass analyzer and provides a method of identifying the presence of and quantifying the abundance of analytes in liquid samples. In a preferred embodiment, the droplet production axis is coaxial with the centerline of the mass analyzer to provide optimal ion transmission efficiency. In this embodiment, the output of the ion source is drawn into a mass analyzer to determine the mass to charge ration (m/z) of the ions generated from charged droplets generated by the droplet source of the present invention.
In an exemplary embodiment, the ion source of the present invention is coupled to an orthogonal time of flight (TOF) mass spectrometer to provide accurate measurement of m/z for compounds with molecular masses ranging from about 1 amu to about 50,000 amu. In a more preferred embodiment, pulsed droplet formation is synchronized with the extraction pulse of the TOF mass spectrometer. Synchronization of droplet production events and ion detection via pulsed orthogonal extraction is beneficial because it provides a detection efficiency (detection efficiency=(ions detected)/(ion formed)) independent of the duty cycle of the TOF mass analyzer. Other exemplary embodiments include, but are not limited to, ion sources of this invention operationally coupled to quadrupole mass spectrometers, tandem mass spectrometers, ion traps or combinations of these mass analyzers.
In an exemplary embodiment, the ion source of the present invention is coupled with a mass spectrometer to provide a method of single droplet mass spectrometry. In this embodiment, a mass spectrum is obtained for each individual droplet formed by the piezoelectric element.
Alternatively, the ion source of the present invention may be operationally connected to a device capable of classifying and detecting gas phase analyte ions on the basis of electrophoretic mobility. In an exemplary embodiment, the ion source of the present invention is coupled to a differential mobility analyzer (DMA) to provide a determination of the electrophoretic mobility of ions generated from liquid samples. This embodiment is beneficial because it allows ions of the same mass to be distinguished on the basis of their electrophoretic mobility, which in turn depends on the molecular structure of the gas phase ions analyzed.
The present invention also comprises methods of increasing the transmission efficiency of gas phase analyte ions generated by field desorption methods to a mass analyzer region. The ion source of the present invention is capable of generating a stream of gas phase analyte ions with a selectively directed momentum along a droplet production axis and with a substantially uniform trajectory along the droplet production axis. Coaxial alignment of the droplet production axis along the centerline axis of a mass analyzer, such as a time-of-flight detector, provides significant improvement of ion transmission efficiency over conventional ion sources. Enhanced ion transmission efficiency is beneficial because it results in increased sensitivity in the subsequent mass analysis and detection of chemical species.
In a preferred embodiment, the present invention comprises a device to analyze the composition of individual cells. In this embodiment, the liquid sample is prepared by lysing the analyte cell and subsequently separating the biomolecules, such as proteins and DNA, into separate fractions via a suitable liquid phase purification method. Next, the liquid sample is introduced to the dispenser element where it is dispensed into a stream of individual charged droplets or packets of charged droplets. Subsequent field desorption generates a source gas phase analyte ions that is conducted to a charged particle analysis region. In a preferred embodiment, the orthogonal time-of-flight mass spectrometry is used to determine the identity and concentration of biomolecules in the liquid sample prepared from the single cell.
The invention further provides methods of generating charged droplets employing the device configurations described herein. Additionally, the invention provides methods for the analysis of liquid samples, particularly biological samples employing the device configurations described herein.
The invention is further illustrated, but not limited, by the following description, examples and drawings.